Canine erythropoietin gene and recombinant protein

ABSTRACT

One aspect of the present invention is an isolated nucleic acid molecule encoding canine erythropoietin. The present invention also relates to an isolated canine erythropoietin protein or polypeptide. Another aspect of the present invention is a method for providing erythropoietin therapy to a dog or a cat including administering recombinant canine erythropoietin to a dog or a cat in need of erythropoietin therapy is an amount sufficient to increase production or reticulocytes and red blood cells in the dog or cat.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/082,669, filed Apr. 22, 1998.

FIELD OF THE INVENTION

The present invention relates to recombinant canine erythropoietin andits use in methods for providing erythropoietin therapy to a dog or cat.

BACKGROUND OF THE INVENTION

Erythropoietin is a glycosylated protein that stimulates red blood cellproduction. It is produced by interstitial and capillary endotelialcells in the renal cortex and transported in the blood to the bonemarrow. Koury et al., “Localization of Erythropoietin Synthesizing Cellsin Murine Kidneys by in situ Hybridization,” Blood, 71:524-527 (1988);Eschbach, “The Anemia of Chronic Renal Failure: Pathophysiology and theEffects of Recombinant Erythropoietin,” Kidneys Int., 35:134-148(1989).The hormone's biological activity involves a direct receptor-mediatedstimulation of the maturation and replication of late erythroidprogenitor cells, proerythroblasts, and erythroblasts. Mufson et al.,“Binding and Internalization of Recombinant Human Erythropoietin inMurine Erythroid Precursor Cells,” Blood, 69:1485-1490 (1987); Krantz etal., “Specific Binding of Erythropoietin to Spleen Cells Infected withthe Anemia Strain of Friend Virus,” Proc. Natl. Acad. Sci. USA,81:7574-7578 (1984). Synthesis of erythropoietin is stimulated inresponse to tissue hypoxia mediated by intracellular aerobic metabolism.Erslev, “Physiologic Controls of Red Cell Production,” Blood, 10:954-959(1955). The primary protein structure of human erythropoietin includes a27 amino acid signal peptide and a 166 amino acid mature protein. Lin etal., “Cloning and Expression of the Human Erythropoietin Gene,” Proc.Natl. Acad. Sci. USA, 82:7580-7584 (1985). Predicted molecular weight of18.4 kDa is substantially less than the 32-kDa observed whenerythropoietin is purified directly from blood or urine. The differenceis due to glycosylation, three N-linked sugar chains at Asn, 24, 38, and83, and an O-linked mucin-like moiety at Ser 126. Lai et al.,“Structural Characterization of Human Erythropoietin,” J. Biol. Chem.,261:3116-3121 (1986). Compared to human, the amino acid sequences ofmouse and monkey erythropoietin are 80 and 92% identical, respectively.McDonald et al., “Cloning, Sequencing, and Evolutionary Analysis of theMouse Erythropoietin Gene,” Molecular and Cellular Biology, 6:842-848(1986): Shoemaker et al., “Murine Erythropoietin Gene: Cloning,Expression, and Human Gene Homology,” Molecular and Cellular Biology,6:849-858 (1986); Lin et al., “Monkey Erythropoietin Gene: Cloning,Expression and Comparison with the Human Erythropoietin Gene,” Gene,44:201-209 (1986). The basic erythropoietin gene structure, five exonsand four introns, is conserved.

Recombinant human erythropoietin (rhEPO) synthesized in Chinese HamsterOvary (CHO) cells is produced commercially (Epogen®, Amgen, Inc.Thousand Oaks, Calif.) and widely used to support red blood cellproduction in people suffering from anemia secondary to chronic renaldisease. Eschbach, “The Anemia of Chronic Renal Failure: Pathophysiologyand the Effects of Recombinant Erythropoetin.” Kidney Int., 35:134-148(1989); Eschbach et al., “Treatment of the Anemia of Progressive RenalFailure with Recombinant Human Erythropoietin,” N. Engl. J. Med.,321:158-163 (1989). Although the pathogenosis of the anemia ismultifactorial, compensatory failure by the bone marrow to replace redblood cells largely involves a loss of functional renal tissue and adrop in endogenous erythropoietin production. Eschbach, “The Anemia ofChronic Renal Failure: Pathophysiology and the Effects of RecombinantErythropoietin,” Kidney Int., 35:134-148 (1989); King et al., “Anemia ofChronic Renal Failure in Dogs,” J. Vet. Int. Med., 6:264-270 (1992).Synthesis of rhEPO for clinical use in restricted to eukaryotic cellsdue to the requirement of post-translational glycosylation for in vivostability and bioactivity of the hormone. Takeuchi et al., “Structuresand Functional Roles of the Sugar Chains of Human Erythropoietins,”Glycobiology, 1:337-346 (1991). Devoid of sugars or even the terminalsialic acid residues, erythropoietin is rapidly cleared and metabolizedby the liver. Spivak et al., “The in vivo Metabolism of RecombinantHuman Erythropoietin in the Rat,” Blood, 73:90-99 (1989).

Nonregenerative anemia, characterized by an inadequate production of newred blood cells, is a frequent and serious complication of kidneyfailure, certain types of cancer, and other chronic diseases incompanion animals.

Chronic renal failure is a progressive and irreversible deterioration ofkidney function that is a common and frustrating clinical problem inveterinary medicine. Although usually considered a disease of olderanimals, chronic renal failure is also encountered congenitally asfamilial renal disease (e.g., in the Norwegian elkhoud, Cocker spaniel,Samoyed, Doberman pinscher, Lhasa apso, Shih Tzu, golden retriever)(Finco, “Congenital, Inherited and Familial Renal Diseases,” In: Canineand Feline Nephrology and Urology. Osborne et al., (eds.), Baltimore:Williams & Wilkins, pages 471-483 (1995)) and in other young animalsthrough nephrotoxic or infection mechanisms. Polzin et al., “Diseases ofthe Kidneys and Ureters,” In: Textbook of Veterinary Internal Medicine,Ettinger (ed), Philadelphia: WB Saunders Company. pp. 1962-2046 (1989):Krawiec. “Renal Failure in Immature Dogs.” J. Amer. Anim. Hosp. Assoc.23:101-107 (1987). Despite a poor long-term prognosis, many dogs andcats with chronic renal failure are medically managed for years withspecial diets, phosphate binders, and antacids. Eventually, however,this conventional therapy fails to control the clinical signs of renalfailure. Cowgill et al., “Veterinary Applications of Hemodialysis,” In:Kirk's Current Veterinary Therapy, 12th ed., Bonagura et al., (eds.),Philadelphia: W B Saunders, pages 975-977 (1995). For these animals,intermittent hemodialysis has improved survival by decreasing the uremictoxins that accumulate during renal failure. Operational dialysis unitsare already available in several veterinary centers across the country,and expanded use of hemodialysis in the management of renal failure inveterinary medicine is expected.

Nevertheless, even though dialysis ameliorates the uremia in canine andfeline patients, lethargy, weakness, and inappetence resulting from theanemia of chronic renal failure persist. In fact, the anemia may even becompounded by blood loss in the dialyzer. Eschbach et al., “Iron Balancein Hemodialysis Patients,” Ann. Int. Med., 87:710-713 (1977).Erythropoietin treatment has become an essential component of thetherapy for animals receiving hemodialysis. Even with thelife-threatening risk of red cell aplasia, rhEPO is used because itrepresents the only erythropoietin-replacement option currentlyavailable.

Lymphosarcoma (also known as lymphoma or malignant lymphoma) is a commoncancer in dogs. Although the exact cause is unknown, certain breedsincluding Boxer, Basset hound, St. Bernard, Scottish terrier, Airedaleterrier, English bulldog, and Labrador retriever have a predispositionfor development of this cancer. Nelson et al., Essentials of SmallAnimal Internal Medicine. St. Louis: Mosby-Year Book, Inc., pages861-870 (1992). Treatment of lymphosarcoma consists of variouschemotherapy protocols (typically utilizing vincristine,cyclophosphamide, doxorubicin, and prednosone) that result in highremission rates and allow survival for approximately 6-12 months.

Nonregenerative anemia is a common hematologic finding in dogs withlymphosarcoma. Nelson et al., Essentials of Small Animal InternalMedicine, St. Louis: Mosby-Year Book, Inc, pages 861-870 (1992); Lucroy,et al., “Anaemia Associated with Canine Lymphoma,” Comp. Haematol. Int'l8:1-6 (1998). The anemia may be encountered during the initialdiagnostic evaluation, or may develop during chemotherapy. Similarly,human cancer patients are often anemic, Miller et al., “DecreasedErythropoietin Response in Patients with the Anemia of Cancer.” N. Engl.J. Med., 322:1689-1692 (1990); Moliterrio et al., “Anemia of Cancer,”Hematol. Oncol. Clin. of N. Am., 10:345-363, (1996). Although thepathogenesis of the anemia of cancer is multifactorial, three majorvariables identified are: 1) the inhibition of erythropoietin productionand bioactivity by inflammatory cytokines and chemotherapeutic drugs; 2)direct inhibition of erythroid progenitors by cytokines; and 3) impairediron metabolism. Moliterrio et al., “Anemia of Cancer,” Hematol. Oncol.Clin. of N. Am., 10:345-363 (1996); Schapira et al., “SerumErythropoietin Levels in Patients Receiving Intensive Chemotherapy andRadiotherapy,” Blood, 76:2354-2359 (1990); Means et al., “Progress inUnderstanding the Patheogenesis of the Anemia of Chronic Disease,”Blood, 80:1639-1647 (1992); Lacome, “Resistance to Erythropoietin.” N.Eng. J. Med., 334: 660-662 (1996); Beguin, “Erythropoietin and theAnemia of Cancer,” Acta. Clinica. Belgica, 59:36-52 (1996); Mittelman,“Anemia of Cancer: Pathogenesis and Treatment with RecombinantErythropoietin,” Isr. J. Med. Sci., 32:1201-1206 (1996). Consistent withthese etiologic variables is clinical data demonstrating that the anemiaof cancer in 32-85% of human patients (depending on the cancer type)responds to pharmacologic doses of rhEPO. Mittelman, “Anemia of Cancer:Pathogenesis and Treatment with Recombinant Erythropoietin,” Isr. J.Med. Sci., 321201-1206 (1996); Spivak, “Recombinant Human Erythropoietinand the Anemia of Cancer,” Blood, 84:997-1004 (1994); Henry,“Recombinant Human Erythropoietin Treatment of Anemia Cancer Patients,”Cancer Practice, 4:180-184 (1996). Furthermore, in vitro studiesdemonstrate a reversal of cytokine-mediated inhibition of erythropoiesiswith increased concentrations of rhEPO. Means et al., “Inhibition ofHuman Erythroid Colony-Forming Units by Gamma Interferon can beCorrected by Recombinant Human Erythropoietin.” Blood, 78:2564-2567(1991). However, treatment with a safe “non-immunogenic” preparation ofexogenous erythropoietin to alleviate the anemia associated with cancerand chemotherapy in dogs or cats has not been possible.

As noted above, erythropoietin therapy is often indicated for themanagement of nonregenerative anemia. In cases of primary erythropoietindeficiency, as in anemia secondary to chronic renal failure,erythropoietin therapy may become essential for life. The only opinioncurrently available to veterinarians is rhEPO, with its inherent risk ofimmunogenicity. Cowgill, “Erythropoietin: Its Use in the Treatment ofChronic Renal Failure in Dogs and Cats,” Proceedings of the 15th AnnualWaltham/OSU Symposium for the Treatment of Small Animal Diseases. OhioState University, pages 65-71 (1991): Giger, “Erythropoietin and ItsClinical Use,” Compend. Contin. Ed. Pract. Vet., 14:25-34 (1992);Cowgill, “Medical Management of the Anemia of Chronic Renal Failure.”In: Canine and Feline Nephrology and Urology, Osborne, et al., (eds.),Baltimore: Williams and Wilkins, pages 539-554 (1995); Cowgill et al.,“Use of Recombinant Human Erythropoietin for Management of Anemia inDogs and Cats with Renal Failure. J. Am. Vet. Med. Assoc., 212:521-528(1998); Stokol et al., “Pure Red Cell Aplasia After Recombinant HumanErythropoietin Treatment in Normal Beagle Dogs,” Vet. Pathol., 34:474(1997). When dogs develop red cell aplasia secondary to rhEPO, continuedtherapy is contraindicated for two reasons. First, the in vivobioactivity of rhEPO is blocked, most likely because it no longer evenreaches the erythroid progenitor target cells in the bone marrow.Second, the rhEPO therapy is casually associated with the red cellaplasia. Spontaneous recovery of the bone marrow is possible withcessation of the rhEPO treatments. Unfortunately, in many of theclinical cases where either the production or bioactivity of endogenouserythropoietin is compromised by the patient's primary disease, thisspontaneous recovery of erythropoiesis never develops or is soinadequate that the red cell aplasia proves to be fatal.

In dogs and cats, the progressive clinical syndrome associated withchronic diseases, such as renal failure, also includes development of anonregenerative anemia. In parallel to the human literature, studieshave documented low serum concentrations of erythropoietin despite theanemia. King et al., “Anemia of Chronic Renal Failure in Dogs,” J. Vet.Int. Med., 6:264-270 (1992). Therapeutic use of rhEPO in dogs and catswith anemia secondary to chronic renal failure results in a rapid andsignificant red blood cell response. Cowgill, “Erythropoietin: Its Usein the Treatment of Chronic Renal Failure in Dogs and Cats.” Proceedingsof the 15th Annual Waltham/OSU Symposium for the Treatment of SmallAnimal Diseases. Ohio State University, pages 65-71 (1991); Giger,“Erythropoietin and Its Clinical use,” Compend. Cantin. Ed. Pract. Vet.,14:14 25-34 (1992); Cowgill, “Medical Management of the Anemia ofChronic Renal Failure,” In: Canine and Feline Nephrology and Urology.Osborne et al. (eds.). Baltimore: Williams and Wilkins, pages 539-554(1995); Cowgill et al., “Use of Recombinant Human Erythropoietin forManagement of Anemia in Dogs and Cats with Renal Failure.” J. Am. Vet.Med. Assoc., 212:521-528 (1998). Depending on the dose administered,hematocrit and hemoglobin values can be restored to a normal rangewithin several weeks and treated animals display increased alertness,physical strength, appetite, and overall attitude. These findingsstrongly suggest that the persistent anemia contributes significantly tosome of the clinical manifestations of chronic renal failure.Unfortunately, the red blood cell status of both dogs and cats oftendeclines in 1 to 4 months despite continued rhEPO therapy. Therapeuticfailure of rhEPO in companion animals, estimated with an incidencebetween 20 and 50%, appears to result from interspecies variation inerythropoietin structure and the appearance of antibodies against thehuman protein. The ability of rhEPO to bind target receptors onerythroid progenitor cells is conserved, but the human protein isfrequently recognized as foreign by the immune system of animals.Cowgill, “Erythropoietin: Its Use in the Treatment of Chronic RenalFailure in Dogs and Cats,” Proceedings of the 15th Annual Waltham/OSUSymposium for the Treatment of Small Animal Diseases. Ohio StateUniversity, pages 65-71 (1991); Giger, “Erythropoietin and Its ClinicalUse,” Compend. Contin. Edg., Pract. Vet., 14:25-34 (1992); Cowgill,“Medical Management of the Anemia of Chronic Renal Failure,” In: Canineand Feline Nephrology and Urology. Osborne et al., (eds.), Baltimore:Williams and Wilkins, pages 539-554 (1995); Cowgill et al., “Use ofRecombinant Human Erythropoietin for Management of Anemia in Dogs andCats with Renal Failure,” J. Am. Vet. Med. Assoc., 212:521-528 (1998).

Anti-rhEPO antibodies are thought not only to effectively block rhEPO'sbioactivity, but also have the potential to cross react with residualendogenous erythropoietin and lead to a pure red cell aplasia. Thisproblem of immunogenicity can be life threatening and has severelylimited the therapeutic potential of rhEPO for veterinary applications.The concept of erythropoietin replacement is appropriate for comparisonanimals, the problem is the immunogenicity of rhEPO.

The cDNA sequences for a number of mammalian erythropoietin genes aredisclosed in Wen, et al., “Erythropoietin Structure-FunctionRelationships: High Degree of Sequence Homology Among Mammals,” Blood82(5):1507-16 (1993). Although the nucleotide sequence for dog isdisclosed, that sequence is missing the coding information for the firstfour codons of canine erythropoitin, which is critical for recombinantproduction of this protein.

The present invention is directed to overcoming the above-noteddeficiencies in the prior art.

SUMMARY OF THE INVENTION

One aspect of the present invention is an isolated nucleic acid moleculeencoding canine erythropoietin.

The present invention also relates to an isolated canine erythropoietinprotein or polypeptide.

Another aspect of the present invention is a method for providingerythropoietin therapy to a dog including administering recombinantcanine erythropoietin to a dog, in need of erythropoietin therapy in anamount sufficient to increase production of reticuloyctes and red bloodcells in the dog.

The present invention also relates to a method for providingerythropoietin therapy to a cat including administering recombinantcanine erythropoietin to a cat in need of erythropoietin therapy in anamount sufficient to increase production of reticuloyctes and red bloodscells in the cat.

Recombinant canine erythropoietin (“rcEPO”) exhibits comparablebiological activity of rhEPO. Further, through the absence (dog) orsignificant reduction (cat) of the interspecies variation in proteinstructure, rcEPO stimulates erythropoiesis while avoiding theimmunogenicity problems that occur with rhEPO. Thus, the availability ofrcEPO should provide veterinarians and pet owners with a valuabletherapeutic modality to improve the quality of life for dogs and catssuffering from the anemia of chronic renal failure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of a canine EPO (“cEPO”) expressionplasmid. The cEPO gene contained within a 4.4 kb Xba I fragment wasdigested with the restriction enzyme Sgr A1 to remove an ATG located 66bases upstream of the translational start site. Identity of the cEPOtranslational start and stop codons were deduced by comparison topublished erythropoietin sequence data for mouse, monkey, and human. TheSgr A1-Xba I fragment was then blunt ended with S1 nuclease and ligatedinto the BamHI site of the eukaryotic expression vector pLEN that wassimilarly blund ended. In this construct, constitutive transcription ofcEPO is driven by SV40 enhancer and human metallothionein promotersequences.

FIG. 2 shows a comparative analysis of steady state cEPO mRNA in chinesehamster ovary (“CHO”) cell clones. Total RNA was isolated from 100individual CHO cell clones by acid guanidiniumthiocyaante-phenol-chloroform extraction followed by differentialalcohol and salt precipitation. The RNA was resolved electrophoretically(5 μg/lane), transferred to a nylon membrane, and hydridizedsequentially with ³²P-labeled cDNA probes for canine erythropoietin andthe housekeeping gene EFTu. Clones with high levels of cEPO expressionwere identified by phosphor image quantitation (Fujix Bio-imaging andMacBAS software, Fuji (Stamford, Conn.) of steady state cEPO mRNA levelsnormalized to the expression of EFTu.

FIG. 3 shows a Western Blot analysis of rcEPO and rhEPO structure.Conditioned medium (30 μm) from a high rcEPO expressing CHO cell clonewas compared to 15 units of rhEPO both without (−) or with (+)pretreatment with N-glycosidase (PNGase F, New England Biolabs, Beverly,Mass.). The samples were resolved electrophoretically under reducingconditions by SDS-PAGE and transferred to a nitrocellulose membrane. Themembrane was then incubated with a primary antibody againsterythropoictin and developed using a commercial enhancedchemiluminescence procedure (ECL Western Blotting detection system,Amersham, Arlington Heights, Ill.). Control samples were tissue culturemedium (30 μl) conditioned by CHO cells that were not transfected withthe pLEN-cEPO expression plasmid.

FIGS. 4A and 4B show the stimulation of erythroid progenitor celldivision with rcEPO (FIG. 4A—conditioned medium from either control orrcEPO-expressing CHO cells) and rhEPO (FIG. 4B—rhEPO (Epogen®, Amgen,Thousand Oaks, Calif.). Extra-medullary hematopoiesis was stimulated ina mouse by phenylhydrazine-induced intravascular hemolysis. Erythroidprogenitor cells were then isolated from the spleen and cultured for 22hours in the presence of increasing concentrations of erythropoietin.The erythroid cell cultures were pulsed with 0.2 μCi ³H-thymidine duringthe last two hours of incubation. Cellular replication was evaluated by³H-thymidine incorporation into newly synthesized DNA. Data pointsrepresent the mean (+/− standard deviation) of each concentrationanalyzed in triplicate.

FIG. 5 shows the stimulation of reticulocytosis in mice with rcEPO andrhEPO. Normal C57BL/6J mice (approximately 8 weeks of age) were injectedsubcutaneously for three days in succession with rcEPO or rhEPO(Epogen®, Amgen, Thousand Oaks, Calif.) in a total volume of 200 μl PBS.Quantitative estimates of rcEPO units were based on in vitro bioactivityand Western Blot analyses. Control mice received injections of culturemedium conditioned by nontransfected CHO cells. One day after the thirdinjection, the mice were sacrificed and peripheral blood collected intoEDTA-containing tubes. The percent of reticuloyctes in each blood samplewas determined by flow cytometric analyses of 10,000 cells using thefluorescent dye thiazole orange (Retic-COUNT, Bectin-Dickinson). Eachgroup represents the mean reticulocyte count (+/− standard deviation) of4 mice. Different letters indicate p<0.05 between treatment groups.

FIGS. 6A and 6B show the hematocrit and reticulocyte response to humanor canine recombinant erythropoietin in normal Beagles. In the firstfour weeks, both dogs were treated with diluent. Starting with weekfour, either rhEPO (FIG. 6A) or rcEPO (FIG. 6B) was administeredsubcutaneously at a dose of 100 units/kg thrice weekly. Changes inweekly hematocrit (▪) and reticulocyte (o) values are illustrated.

FIGS. 7A and 7B show pooled data illustrating hematocrit (FIG. 7A) andreticulocyte (FIG. 7B) response to human or canine recombinanterythropoietin in normal Beagles. During the first four weeks, dogs inboth groups were treated with diluent. Starting with week four, eitherrcEPO (▪, n=7) or rhEPO (o, n=6) was administered subcutaneously andthrice weekly at a dose of 100 units/kg. Changes in the weeklyhematocrit and reticulocyte values are illustrated. Therapy was haltedin all rhEPO-traeted dogs during the experimental period due todevelopment of red cell aplasia. Administration frequency of rcEPO wasreduced in some dogs due to hematocrit values that rose above 65%.

FIG. 8 demonstrates that recombinant cEPO rescues a dog fromrhEPO-Induced red cell aplasia. Hematocrit is plotted over time. Forapproximately 12 weeks rhEPO was administered to the dog three times perweek, followed by 12 weeks of administrationn of rcEPO, again at threetimes per week.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is an isolated nucleic acid moleculeencoding canine erythropoietin. The present invention provides the fulllength genomic sequence of the canine erythropoietin gene.

In a preferred embodiment, the isolated nucleic acid molecule is capableof being expressed in transfected cells and which hybridize to a nucleicacid molecule having a nucleotide sequence of SEQ ID No. 1 understringent conditions but not to a nucleic acid molecule encoding humanerythropoietin under identical conditions.

The DNA molecule which encodes rcEPO comprises the nucleotide sequencecorresponding to SEQ ID No. 1 as follows (codons are given as triplicatenucleotides and noncoding introls and flanking regions are given ascontinuous nucleotides):

TCTAGAACAAGTACTGGGATTGCGAGAAGGAAGGCAACTTGCCTCTGCCCGCACCTTCCCGGCTTCCAAGGCTAGTTGCCCCGCAGGCACCAGGCACCGGCGCTCCCAGCTCGATCCCCCGCCCAGGACTGGGACGCACCCCTCCCCCCGGGGGGAGGGGGGCGGGAGCCTCGGGGTCCCCGGCCTTTCCCAGAATGGCACCCCTCCCGCGGGTGCGCACCCAGCCGCGCCTCCCACAACCCGGGGTCAGACTGGCGGACCCGCGTCCCGCTCCGCGCCTGCTGCCGCGCCTGCTGCCGCTCTGCTCCCGCCCCGGCGAGCCCCCGACCCAGGCGTCCTCCCCCGGTCTGACCCCTCTGGCCCTTACCTCTGGCGACCCCTCACGCACACAGCCTGCCCCCCACCCCCACACACGCACGCACACATGCTGATAACAGCCCCGACCCCCGGCCGAGCCGCAGTCCCCGGGCCAACCCCGGCCGGTCGCCGCGCGCCTGTCCTCGCGGACCCTGGCCGAGAGCCCTGCGCTCGCTCTGCGCGACCCCGGCTCGGCGGCCCCTGGACGGTGGCCCCCTCCTTCGGACCGTGGGGCCGGCCCTGCCCCGCCGCGCTTCCCGGGATGAGGGCTCCCGGCGAGGGCGCCGGCGGAGCCCCTGGTCGCTGAGCGGCCGACGGAGGCGCGGAG ATG GGG GCG TGC GGTGAGTACTCGCCGGCCGGAGGAGCCCCCGCCCGCTCGGGCCCCTGTTTGAGCAAGAATTTACCGCTGGGGCCCCGAGGTGGCTGGGTTCAAGGACCGACGACTTGCCAAGGACCCCGGAAGGGCAAGGGGGGTGGCCCAGCCCCCACGTGCCGGCAGGGCTTAGGGAGCCCCTAGGAAAGGTGAAATCTGACCTGGACACGGGGATGCCGTTTGGGGGTTCAGGGAGCCGAGGGGCTGCCACGTGCGTGGGGAGAAGGCTGATACCTGGGTCTTGGAGCAATCACCGGGATCTGCCAGAGGGGAAGCCTCAGTCACGCCGGGATTGAAGTTTGGCCGGGAGAAGTGGATGCCGGTAGTTTGGGGGGTGGGCTGTGCGCGCAGCAGCGGCCGGATTGAATGAAGGCAGGGCAGGCAGAACCTGAACGCTGGGAAGGTGGGGGTCGGGCGCGACTAGTTGGGGGCAGAGGAGCGGGATGTGTGAACCTGCCCCTCCAAACCCACACAGTCAGCCTGGCACTCTTTTCCAG AA TGT CCT GCC CTG TTCCTT TTG CTG TCT TTG CTG CTG CTT CCT CTG GGC CTC CCA GTC CTG GGC GCC CCCCCT CGC CTC ATT TGT GAC AGC CGG GTC CTG GAG AGA TAC ATC CTG GAG GCC AGGGAG GCC GAA AAT GTC ACGGTGAGGGTCCCACCTCAGGACATTCTCAGTAGTCCAGGGGTGTCCTCCAAGATCTGGGAACCTGAGCCCCTTCGTTCAGAGATGGAGATGGGAAGCCAGAGCCCTCAGGAAAAATGATAAAAGTGGTAGCCCCTCAATGCATGCCTGGAAGCTAGATCAGGGGCAAAGCTGGAGGGAGCTCTTGGGGAGCCTGACACCCCCTTCCCCCCGACCTGGGCTCATGCATTTCAG ATG GGC TGT GCT CAAGGC TGC AGC TTC AGT GAG AAT ATC ACC GTC CCA GAC ACC AAG GTT AAT TTC TATACC TGG AAG AGG ATG GATGTGAGTTTATTTTTCCCCTCTACTTGGACAGTCTTGTTTTGCTTACCTGATGGGGTGGGAGGGAGTACCATAGAAGAAGCTGAGGGCTCAATGCAATATGTTTACTCATTTGTTCTTTGTTCATTCATTAATTCATTCATTCAATGAAACTGATTCCAAGCCTTCATTTTGCTCAGCTTGGTGCTTGGGGCTGCTGAGAGGGAGGGGCTGGCCTGGGCCGCTGACTATAAGTCGCCATTC CCTTTAG GTTGGG CAG CAG GCC TTG GAA GTC TGG CAG GGC CTG GCA CTG CTC TCA GAA GCC ATCCTG CGG GGT CAG GCC CTG TTG GCC AAC GCC TCC CAG CCA TCT GAG ACT CCG CAGCTG CAT GTG GAC AAA GCC GTC AGC AGC CTG CGC AGC CTC ACC TCT CTG CTT CGGGCG CTG GGA GCC CAGGTGGGTAGAAGCCTCCCTTGCACTTCTGCTCCAAGGGCCCTGCCAAGAAATACTGAGACCCCACTGGACCTCCTCATCCCCCCTCCAATTCTGTCCTCCATCCCATCTCCCACCAGGGTCCTGGGCACTTCGGTAACCTTCTCTTCTCTCCTTGTCAG AAG GAG GCC ATG TCC CTT CCA GAGGAA GCC TCT CCT GCT CCA CTC CGA ACA TTC ACT GTT GAT ACT TTG TGC AAA CTTTTC CGA ATC TAC TCC AAT TTC CTC CGT GGA AAG CTG ACA CTG TAC ACA GGG GAGGCC TGA AGA AGA GGA GAC AGG TGACCAGGTGCTCCCACCCCAGGCACATCCACCACCTCACTCACTACCACTGCCTGGGCCACGCCTCTGCACCACCACTCCTGACCCCTGTCCAGGGGTGATCTGCTCAGCACCACCCTGTCCCTTGGACACTCCACGGCCAGTGGTGATATCTCAAGGGCCAGAGGAACTGTCCAGAGCTCAAATCAGATCTAAGGATGTCACAGTGCCAGCCTGAGGCCCGAAGCAGGAGGAATTCGGAGGAAATCAGCTCAAACTTGGGGACAGAGCCTTGCTCGGGAGACTCACCTCGGTGCCCTGCCGAACAGTGATGCCAGGACAAGCTGGAGGGCAATTGCCGATTTTTTGCACCTATCAGGGAGAGACAGGAGAGGCTAGAGAATCTAGGTGGCAAGCCATAAATTCTCTAGGTCTCGTGGGTCTCCTATGACAGCAAGAGCCCACTGGCAAAGGGTGGTGGCAGCCATGGAGATGGGATAGGGGCTGGCCCCTGGCTCTCATTGGGTCCAAGTTTTGTGTATTTTTCAATCTCATTGGCAGAAACTGAAACCACAACATGGCTCTTGACTTTTCTGTTTTCCCTGGGATCCTCCTACTTCCCTGGCCCTGCTCCGGCCCTGGCAGCAGGCCACAGTCCTGGAAAACTAGAGGTGGAGGGGGTCGGCCCTACGTGCTGCCTCTCATGGTCTATCTGACCTCTTGACCCCACTGGGTCTGAGGCCACAAGCTCTGCCCACGCTGGTCAATAAGGTGGTTCTATTCAAGGCTGTTCCTCAGTAGGCAGTTGGCAACCCTCTGTAGTGAGCTACAGCTGCCATCAAGGAAACAGGAGCCAGGAGGAAGAGCCCCTTTGGGGGCTGGTGGGAGTTCCCAGTCCTGGACCCTGGACCCTTATTATTTCTCACTTCTCCATAGTGCTTTTGACTAAAGCCACATTCCCACATCAGCCTTTGCCACCTCTAAATCCAGCTGACCCTTTTCCTTGCCTGAGGATGGTCAAGGCAAGGAAATGCTCTACCCCAAAACTTGCAGAAGGAGCCACGTTCCCCAAAAGCGGTCTCACTGAGCACTCACTCTGTGCCCAGGGCTATTCTAGGTGGTTCACTTACATGACATTTTATTCCTTGCACAGCCTGATGAGAAAGTTTCCACTGTCATTCCCAGATGAGAAGTAAACTGCCCAAAGCCAAGACAACAGGAATCCCCAATGGCCCCAGCTCTTATCCCTTCCCTCTTCAGCTTATTCTTCCACATAACCCCTACCTGCTCCCTGCTCCCCTGGGATGGGAGACACAGAACAGACTAACTCAGCTCCCGCTCTCCATCCCTACTAATAATTTTACCCAGTACTCCAACATTCCACTTCAAATTCCTTCCCAGAGGGATGCCTTGGTGGCTCAGTGGTAGAGTGTCTGCCTTTGCTCAGGTCGTGATCCCCAGGTCCAGAGATTGAGTCCTGCATCAGGCTCCCTGCAGAGAGCCCAATCCTGCCATTATCATATGTGTGGGGATCAGCCTTTCTGCTCATATCACAAAACTTAGAGAAGTCAGCCTGCATCCCTGAAAATATCAAAAGAAAAAGAATTTTTGCAATCTGCAGGAGGACAAATGATGGGTCGGTTGGGGGATTGGATGGTATGTGCTAAATATATGTGTGTGTGCTGGGGGGCCGTGCCAAGCGTGGTGGGAGGAATCAAAGGAGAGGTGGACCCAAAGGAGAATTCCCCCCTCCTCCCCTGCCTGGCCAACTCAGTTCCTAGGGTATACTGCCCTCTTCAGGCCCCACTGGAAAATGTTAGAGAAATACACAAGTCAAAGAGCCCTTAGGTCTCTGATTATTCTTTGCACATTTCAATAAAAATTTGTATTACAGTTTCCACAGATGGCATCTGGTTCTTGCCCCACTGCTGTGAAACAGTAAGGGAGGAATCTGTCTCTCTCGCTGNCAAAATCGAAGCTAAGAGAGGTGTCCAAGGCATGCAGCTAATAATGGTAGCTAGGACCTGAACACAAGGTTTAGGAATCGTAACCTCCAAGCCCATCTTAGCCTGATGTGTCATCTAGA

!

In a most preferred embodiment, the nucleic acid molecule comprises thenucleotide sequence of SEQ ID No. 1.

The canine erythropoietin cDNA sequence is given as SEQ. ID. No. 2, asfollows:

ATG GGG GCG TGC GAA TGT CCT GCC CTG TTC CTT TTG CTG TCT TTG CTG CTG CTTCCT CTG GGC CTC CCA GTC CTG GGC GCC CCC CCT CGC CTC ATT TGT GAC AGC CGGGTC CTG GAG AGA TAC ATC CTG GAG GCC AGG GAG GCC GAA AAT GTC ACG ATG GGCTGT GCT CAA GGC TGC AGC TTC AGT GAG AAT ATC ACC GTC CCA GAC ACC AAG GTTAAT TTC TAT ACC TGG AAG AGG ATG GAT GTT GGG CAG CAG GCC TTG CAA GTC TGGCAG GGC CTG GCA CTG CTC TCA GAA GCC ATC CTG CGG GGT CAG GCC CTG TTG GCCAAC GCC TCC CAG CCA TCT GAG ACT CCG CAG CTG CAT GTG GAC AAA GCC GTC AGCAGC CTG CGC AGC CTC ACC TCT CTG CTT CGG GCG CTG GGA GCC CAG AAG GAG GCCATG TCC CTT CCA GAG GAA GCC TCT CCT GCT CCA CTC CGA ACA TTC ACT GTT GATACT TTG TGC AAA CTT TTC CGA ATC TAC TCC AAT TTC CTC CGT GGA AAG CTG ACACTG TAC ACA GGG GAG GCC TGC AGA AGA GGA GAC AGG TGA

In a preferred embodiment, the nucleic acid molecule encodes the aminoacid sequence of SEQ ID No. 3 as follows (shown under the cDNa openreading frame sequence):

Met Gly Ala Cys Glu Cys Pro Ala Leu Phe Leu Leu Leu Ser< Leu Leu Leu LeuPro Leu Gly Leu Pro Val Leu Gly Ala Pro< Pro Arg Leu Ile Cys Asp Ser ArgVal Leu Glu Arg Tyr Ile< Leu Glu Ala Arg Glu Ala Glu Asn Val Thr Met GlyCys Ala< Gln Gly Cys Ser Phe Ser Glu Asn Ile Thr Val Pro Asp Thr< LysVal Asn Phe Tyr Thr Trp Lys Arg Met Asp Val Gly Gln< Gln Ala Leu Glu ValTrp Gln Gly Leu Ala Leu Leu Ser Glu< Ala Ile Leu Arg Gly Gln Ala Leu LeuAla Asn Ala Ser Gln< Pro Ser Glu Thr Pro Gln Leu His Val Asp Lys Ala ValSer< Ser Leu Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu Gly Ala< Gln LysGlu Ala Met Ser Leu Pro Glu Glu Ala Ser Pro Ala< Pro Leu Arg Thr Phe ThrVal Asp Thr Leu Cys Lys Leu Phe< Arg Ile Tyr Ser Asn Phe Leu Arg Gly LysLeu Thr Leu Tyr< Thr Gly Glu Ala Cys Arg Arg Gly Asp Arg

Suitable nucleic acid molecules include those nucleic acid moleculesencoding an amino acid of a protein or polypeptide sufficiently toduplicative of canine erythropoietin and having a nucleotide sequencewhich is at least 95% homologous and preferably 98% homologous to thenucleotide sequence of canine erythropoietin (“EPO”) (as shown in SEQ IDNo. 1).

While the nucleotide sequence is at least 95% homologous as determinedby the TBLAST Program (Altschul. S. F., et al., “Basic Local AlignmentSearch Tool,” J. Mol. Biol. 215:403-410 (1990), which is herebyincorporated by reference) using the default parameters, nucleotideidentity is not required. As should be readily apparent to those skilledin the art, various nucleotide substitutions are possible which aresilent mutations (i.e. the amino acid encoded by the particular codondoes not change). It is also possible to substitute a nucleotide whichalters the amino acid encoded by a particular codon, where the aminoacid substituted is a conservative substitution (i.e. amino acid“homology” is conserved). It is also possible to have minor nucleotideand/or amino acid additions, deletions, and/or substitutions in thecanine EPO nucleotide and/or amino acid sequences which do not alter thefunction of the resulting canine EPO.

Alternatively, suitable DNA sequences may be identified by hybridizationto SEQ ID No. 1 under stringent conditions. Preferably, suitablesequences would hybridize to SEQ ID Nos 1 under highly stringentconditions where a nucleic acid encoding human EPO would not hybridize.For example, sequences can be isolated that hybridize to a DNA moleculecomprising a nucleotide sequence of 50 continuous bases of SEQ ID No. 1under stringent conditions characterized by a hybridization buffercomprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 65° C.and remaining) bound when subject to washing with the SSC buffer at 65°C.; and preferably in a hybridization buffer comprising 20% formamide in0.9M saline/0.09M SSC buffer at a temperature of 75° C., and remainingbound when subject to washing at 42° C. with 0.2×SSC buffer at 75° C.

The DNA molecule encoding the canine EPO protein or polypeptide of thepresent invention can be incorporated in cells using conventionalrecombinant DNA technology. Generally, this involves inserting the DNAmolecule into an expression system to which the DNA molecule isheterologous (i.e. not normally present). The heterologous DNA moleculeis inserted into the expression system or vector in proper senseorientation and correct reading frame. The vector contains the necessaryelements for the transcription and translation of the insertedprotein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference, describes the production of expression systems in the formof recombinant plasmids using restriction enzyme cleavage and ligationwith DNA ligase. These recombinant plasmids are then introduced by meansof transformation and replicated in unicellular cultures includingeukaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vacciniavirus. Recombinant viruses can be generated by transfection of plasmidsinto cells infected with virus.

Suitable vectors include, but are not limited to pLEN, the pCDN series(Invitrogen), pRc/CMV2 (Invitrogen), and pNeoEGFP (Clontech), thefollowing viral vectors such as lambda vector system gt11, gt WES.tb,Charon 4, and plasmid vectors such as pBR322, pBR325, ACTC177, pACY1084,pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40,pBluescript II SK +/− or KS +/− (see “Statagene Cloning Systems” Catalog(1993) from statagene, La Jolla, Calif. whic is hereby incorporated byreference), pQE, pIH821, pGEx, pET series (see Studier et al., “Use ofT7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene ExpressionTechnology, vol. 185 (1990), which is herein incorporated by reference),and any derivatives thereof. In a preferred embodiment, the vector iseukaryotic expression vector pLEN. Recombinant molecules can beintroduced into cells via transformation, transduction, conjugation,mobilization, or electroporation. The DNA sequences are cloned into thevector using standard cloning procedures in the art, as described byManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringsLaboratory, Cold Springs Harbor, N.Y. (1982), which is herebyincorporated by reference.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cells used. Host-vector systems include but arenot limited to the following: subcloning a eukaryotic expression vectorinto mammalian cell systems; microorganisms such as yeast containingyeast vectors, mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); and insect cell systems infected withvirus (e.g., baculovirus). The expression elements of these vectors varyin their strength and specification. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eukaryotic promotersdiffer from those of procalyotic promoters. Furthermore, eukaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a procaryotic system, and, further, procaryoticpromoters are not recognized and do not function in eukaryotic cells.Similarly, translation of mRNA in prokaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eukaryotes.For a review on maximizing gene expression, see Roberts and Lauer,Methods in Enzymology, 68:473 (1979), which is hereby incorporated byreference.

Promoters vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gear, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in chinese hamster ovary cells,human metallothionein IIA promoter the regulatory sequences from CMV,RSV or SV40, and the like, may be used to direct high levels oftranscription of adjacent DNA segments. Additionally, promoters producedrecombinant DNA or other synthetioc DNA techniques may be used toprovide the transcription of the inserted gene.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals.

Once the isolated DNA molecules encoding the canine EPO proteins orpolypeptides, as described above, have been cloned into an expressionsystem, they are ready to be incorporated into a host cell. Suchincorporation can be carried out by the various forms of transformationnoted above, depending upon the vector/host cell system. Suitable hostcells include, but are not limited to, mammalian cells (chinese hamsterovary cells, and the like), yeast cells, and insect cells.

The present invention also relates to an isolated canine erythropoietinprotein or polypeptide.

In a preferred embodiment, the protein or polypeptide is sufficientlyduplicative of canine erythropoietin to have the biological property ofcausing bone marrow cells to increase production of reticuloyctes andred blood cells and to have the immunological property of not provokingan immune response in a dog.

Preferably, the protein or polypeptide has an amino acid sequence of SEQID No. 3.

Fragments of the above polypeptides or proteins are also encompassed bythe present invention.

Suitable fragments can be produced by several means. In the first,subclones of the gene encoding the protein of the present invention areproduced by conventional molecular genetic manipulation by subcloninggene fragments. The subclones then are expressed in vitro or in vivo inbacterial cells to yield a smaller protein or peptide that can be testedfor activity according to the procedures described below.

As an alternative, fragments of replication proteins can be produced bydigestion of a full-length replication protein with proteolytic enzymeslike chymotrypsin or Staphylococcus proteinase A, or trypsin. Differentproteolytic enzymes are likely to cleave replication proteins atdifferent sites based on the amino acid sequence of the protein. Some ofthe fragments that result from proteolysis may be active.

In another approach, based on knowledge of the primary structure of theprotein, fragments of a replication protein gene may be synthesized byusing the PCR technique together with specific sets of primers chosen torepresent particular portions of the protein. These then would be clonedinto an appropriate vector for increased expression of a truncatedpeptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences of replicationproteins being produced. Alternatively, subjecting a full lengthreplication protein to high temperatures and pressures will producefragments. These fragments can then be separated by conventionalprocedures (e.g., chromatography, SDS-PAGE).

Variants may also (or alternatively) by modified by, for example, thedeletion or addition of nucleotides that have minimal influence on theproperties, secondary structure and hydropathic nature of the encodedpolypeptide. For example, the nucleotides encoding a polypeptide may beconjugated to a signal (or leader) sequence at the N-terminal end of theprotein which co-translationally or post-translationally directstransfer of the protein. The nucleotide sequence may also be altered sothat the encoded polypeptide is conjugated to a linker or other sequencefor ease of synthesis, purification, or identification of thepolypeptide.

The protein or polypeptide of the present invention is preferablyproduced in purified form (preferably, at least about 80%, morepreferably 90%, pure) by conventional techniques. Published methods thathave been used to purify human erythropoietin from the urine or sicklecell anemia patients should be applicable to the purification of rcEPOfrom conditioned tissue culture medium. (Miyake et al., “Purification ofHuman Erythropoictin,” J. Biol. Chem., 252:5558-5564 (1977); Krystal etal., “Purification of Human Erythropoietin to Homogeneity by a RapidFive-Step Procedure,” Blood, 67:71-79 (1986), which are herebyincorporated by reference).

In a preferred embodiment, the protein or polypeptide of the presentinvention is administered in a pharmaceutical composition including aneffective amount of the protein or polypeptide of the present inventionand a pharmaceutically acceptable diluent, adjuvant, or carrier, asdisclosed below.

In a most preferred embodiment, the pharamceutical composition isadministered in an effective amount to provide erythropoietin therapy toa dog or cat.

Another aspect of the present invention is a method for providingerythropoietin therapy to a dog including administering recombinantcanine erythropoietin to a dog in need of erythropoietin therapy in anamount sufficient to increase production of reticuloyctes and red bloodcells in the dog.

In a preferred embodiment, the dog is suffering from anemia, chronic oracute renal failure. In a most preferred embodiment, the dog issuffering from chronic or acute renal failure and the dog is one of thefollowing breeds: Norwegian elkhound, Cocker spaniel, Samoyed, Dobermanpinsher, Lhasa apso, Shih Tsu or golden retriever.

In another preferred embodiment, the dog is suffering from cancer. In amore preferred embodiment, the dog is suffering from lymphosarcoma andthe dog is one of the following breeds. Boxer, Basset hound, St.Bernard, Scottish terrier, Airedal terrier, English bulldog, andLabrador retriever.

In another embodiment, the dog is suffering from rhEPO induced red cellaplasia.

In another embodiment, the dog is administered recombinant canineerythropoietin prior to undergoing surgery.

Preferably, the recombinant canine erythropoietil (“rcEPO”) of thepresent invention is encoded by a nucleic acid molecule having anucleotide sequence of SEQ ID No. 1.

Preferably, from about 50 units/kg to about 500 units/kg of rcEPO isadministered to the dog to increase production of reticuloyctes and redblood cells in the dog. Most preferably, from about 100 units/kg toabout 200 units/kg of rcEPO is administered to the dog.

The rcEPO of the present invention can be administered orally,parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, intraocularly, intraarterially,intralesionally, or by application to mucous membranes, such as, that ofthe nose, throat, and bronchial tubes. It may be administered alone orwith pharmaceutically or physiologcially acceptable carriers excipients,or stabilizers, and can be in solid or liquid form such as, tablets,capsules, powders, solutions, suspensions, or emulsions.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing thercEPO of the present invention and a carrier, for example, lubricantsand inert fillers such as, lactose, sucrose, or cornstarch. In anotherembodiment, these compounds are tableted with conventional tablet basessuch as lactose, sucrose, or cornstarch in combination with binders likeacacia, cornstarch, or gelatin, disintegrating agents, such ascornstarch, potato starch, or alginic acid, and a lubricant, likestearic acid or magnesium stearate.

The rcEPO of the present invention may also be administered ininjectable dosages by solution or suspension of these materials in aphysiologically acceptable diluent with a pharmaceutical carrier. Suchcarriers include sterile liquids, such as water and oils, with orwithout the addition of a surfactant and other pharmaceutically andphysiologically acceptable carrier, including adjuvants, excipients orstabilizers. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols, such as propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly, for injectionsolutions.

For use as aerosols, the rcEPO of the present invention in solution orsuspension may be packaged in a pressurized aerosol container togetherwith suitable propellants, for example, hydrocarbon propellants likepropane, butane, or isobutane with conventional adjuvants. The materialsof the present invention also may be administered in a non-pressurizedform such as in a nebulizer or atomizer.

The present invention also relates to a method for providingerythropoietin therapy to a cat including administering recombinantcanine erythropoietin to a cat in need of erythropoietin therapy in anamount sufficient to increase production of reticuloyctes and red bloodcells in the cat.

In a preferred embodiment, the cat is suffering from anemia secondary tochronic or acute renal failure, caiicer, or red cell aplasia. In a mostpreferred embodiment, the cat is suffering from lymphosarcoma.

In another embodiment, the cat is administered recombinant canineerythropoietin prior to undergoing surgery.

Preferably, the rcEPO is administered to the cat in the same dosage asdescribed above for dogs.

EXAMPLES Example 1 Isolation and Cloning of Canine Erythropoictin(rcEPO)

The gene encoding canine erythropoietin (cEPO) was isolated from aLambda DASH genomic library of canine DAN partially digested with Sau3A1 (Stratagene, La Jolla, Calif.). Approximately one millionbacteriophage plaques were screened with a 180 base pair cDNA fragmentfrom exon 4 of cEPO. The cDNA fragment was generated by polymerase chainredaction (PCR) amplification of canine (genomic DNA using5′-GTTGGGCAGCAGGCCTTGGAAGT (sense) (SEQ ID No. 4) and5′-CTGGGCTCCCAGCGCCCGAA (antisense) (SEQ ID No. 5) as primers. Theprimers correspond to bases 232-254 and 392-411 of the partial cEPO cDNAavailable through GenBank accession number L13027 and published by Wenet al., “Erythropoietin Structure-Function Relationships: High Degree ofSequence Homology Among Mammals,” Blood, 82:1507-1516 (1993), which ishereby incorporated by reference) The exon 4 fragment was then subclonedinto pGEM-3Zf(+) (Promega, Madison, Wis.), amplified in E. coli strainJM 109 (Promega, Madison, Wis.), re-isolated in large amounts bypreparative restriction digests and agarose gel purification, labeledwith ³²p-dCTP using random hexanucleotide primers (Prime-a-Gene,Promega, Madison, Wis.), and purified by G-50 Sephadex spin columnchromatography (Boehringer Mannheim, Indianapolis, Ind.). A total of 9genomic clones that hybridized specifically to the exon 4 probe wereisolated and plaque purified. Southern blot analyses determined that 8of these clones contained the entire cEPO coding region within a 4.5 kbXba I fragment.

An expression plasmid was constructed by subcloning the cEPO gene(extending from an Sgr A1 site located 40 bases upstream of thetranslational start site to the 3′ Xba I site located 2,060 basesdownstream of the stop codon) into the Bam H1 site of the eukaryoticexpression vector pLEN. Friedman et al., “High Expression in MammalianCells Without Amplification,” Bio/Technology, 7:359-362 (1989), which ishereby incorporated by reference. Identity of the start and stop codonsin the cEPO gene were deduced by comparison to published erythropoictinsequence data for mouse, monkey, and human. Lin et al., “Cloning andExpression of the Human Erythropoietin Gene,” Proc. Natl. Acad. Sci.USA, 82:7580-7584 (1985); McDonald et al., “Cloning, Sequencing, andEvolutionary Analysis of the Mouse Erythropoietin Gene,” Molecular andCellular Biology, 6:842-848 (1986); Shoemaker et al., “MurineErythropoietin Gene: Cloning, Expressions and Human Gene Homology,”Molecular and Cellular Biology, 6:849-858 (1986); Lin et al., “MonkeyErythropoietin Gene: Cloning, Expression and Comparison with the HumanErythropoietin Gene,” Gene, 44:201-209 (1986), which are herebyincorporated by reference. The cEPO aenomic insert and pLEN vector wereboth blunt ended with S 1 nuclease prior to ligation. Orientation ofrecombinants were determined by direct DNA sequence analysis.Constitutive high level transcription of cEPO in this construct wasdriven by the SV40 enhancer and human metallothionein 11A promoter ofpLEN (FIG. 1).

Example 2 Expression of rcEPO and RNA Gel Analyses

A cell culture system for the production of rcEPO was established bycotransfection of the pLEN-cEPO construct with pRSVneo at a 10:1 molarratio using calcium phosphate coprecipitation into Chinese Hamster Ovarycells (CHO-KI, American Type Culture Collection, Rockville, Md.). Gormanet al., “High Efficiency DNA Mediated Transformation of Primate Cells,Sceince, 221:551-553 (1983); Graham et al., “A New Technique for theAssay of Infectivity of Human Adenovirus 5 DNA,” Virology, 52:456-467(1973), which are hereby incorporated by reference. The cells weremaintained at 370° C., 5% CO₂/95% air in Dulbecco's modified Eagle'smedium (Gibco BRL, Grand Island, N.Y., catalog no. 11,965) supplementedwith 10% (v/v) fetal bovine serum. Following transfection, G418 (400μg/ml) was added to the culture medium to eliminate nontransformants. Atotal of 122 transformed CHO cell clones were individually isolated andexpanded. Relative expression of cEPO in each CHO clone was compared inparallel on a transcriptional level by Nothern blot analyses.(Subsequently, CHO-rcEPO cell lines were adapted to grow in serum freemedia, greatly simplifying the biochemical purification procedures ofrcEPO. This expression system routinely achieved media concentration ofrcEPO that exceeded 100 U/ml. Using roller bottles, 20,000 units ofrcEPO can be synthesized in a single culture vessel every 24 hours, anamount sufficient to treat a 10 kg dog for two months). Total RNA wasisolated by acid guanidinium thiocyanate-phenol-chloroform extractionfollowed by differential alcohol and salt precipitations and quantifiedspectrophotometrically. Chomczynski et al., “Single-Step Method of RNAIsolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction,”Anal. Biochem., 162:156-159 (1987), which is hereby incorporated byreference. Each RNA sample (5 μg) was electrophoretically separatedthrough 1.5% agarose, 6.5% formaldehyde, submersed stab gels in buffer(pH 7.0) containing 40 mM MOPS, 10 mM sodium acetate, and 1 mM EDTA. Theseparated RNAs were then transferred to nylon membranes (Magna Charge,Micron Separations, Inc., Westboro, Mass.) by standard capillaryblotting techniques and probed with ³²P labeled cEPO exon 4 cDNAfragment. Sambrook et al., “Molecular Cloning: A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989),which is hereby incorporated by reference. Relative comparisons ofsteady state cEPO and mRNA between individual CHO cell clones weredetermined by direct quantitation of ³²P decay events from thehybridization membrane (Phosphor Imager, Fuji Bio-Imaging, Stamford,Conn.). These data were then normalized to expression of thehousekeeping gene elongation factor Tu (EFTTu) to correct for anyvariation in RNA loading or transfer efficiency between samples. Levineet al., “Elonation Factor Tu as a Control Gene for mRNA Analysis of LungDevelopment and Other Differentiation and Growth Regulated Systems,”Nucl. Acids Res., 21:4426 (1993), which is hereby incorporated byreference.

Example 3 Protein Analyses by Immunoblotting

Conditioned medium (30 μl) from a high cEPO expressing CHO cell clonewas resolved by SDS-15% polyacrylamide gel electrophoresis underreducing conditions in parallel with an equal volume of conditionedmedium from control CHO cells and t5 units of rhEPO. Laemmli, “Cleavageof Structural Proteins During the Assembly of the Head of theBacteriophage T4,” Nature, 267:680-685 (1970), which is herebyincorporated by reference. The three samples were analyzed both with andwithout peptide N-glycosidase F digestion (PNGase F, New EnglandBiolabs, Beverly, Mass.). PNGase F reaction buffers and protocols weresupplied by the manufacturer. Following separation, the proteins weretransferred by electroblotting at 33 V and 4° C. overnight to anitrocellulose membrane (Bio-Rad, Hercules, Calif.). The membrane wasblocked for 1 hour at room temperature with 5% nonfat milk in 50 mMTris, 150 mM NaCl buffer (pH 7.4) with 0.05% (v/v) Tween-20 (TBS-Tbuffer). After rinsing with TBS-T buffer, the membrane was incubatedwith a monoclonal antibody to human erythropoietin (Genzyme, Cambridge,Mass.) at a dilution of 1:1000 for 1.5 hours at room temperature. Themembrane was then rinsed again with TBS-T and bound primary antibodydetected with a horseradish peroxidase-linked goat anti-mouse IgG(Sigma, St. Louis, Mo.) at a dilution of 1:8000. The secondary antibodyincubation step was 1.5 hours at room temperature. After a final rinsewith TBS-T, buffer, peroxidase activity was detected bychemiluminescence (ECL Western blotting detection system. Amersham,Arlington Heights, Ill.) and autoradiography.

Example 4 Erythropoietin Bioassays

The biological activity of rcEPO was examined in vitro using splenicerythroid progenitor cells isolated from phenylhydrazine treated mice.Krystal, “A Simple Microassay for Erythropoietin Based on ³H-thymidineIncorporation into Spleen Cells from Phenylhydrazine Treated Mice,” Exp.Hematol., JI: 649-660 (1983), which is hereby incorporated by reference.For each assay, a single mouse was treated with an introperitonealinjection of phenylhydrazine (60 mg/kg body weight) for 2 consecutivedays to induce intravascular hemolysis. The resulting anemia stimulatedextramedullary red blood cell hematopoiesis in the spleen. The mouse wassacrificed 3 days after the second phenylhydrazine injection. The spleenwas removed by dissection, rinsed in sterile 0.01 M phosphate bufferedsaline (PBS, pH 7.4) at 37° C., and placed in a petri dish containing 5ml Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetalbovine serum (Gibco BRL, Grand Island, N.Y.). Splenic cells weredissected from the capsule, and other consecutive tissues by extrusionthrough a wire mesh and aspiration through a 21-gauge needle. Thecellular suspension was then transferred to a polypropylene tube.Residual pieces of tissue debris were allowed to settle over 5 minutesand the supernatant containing dispersed cells was transferred to a newtube. These spleen cells were then pelleted by centrifugation at 1,000g, resuspended in 25 ml of culture medium, counted, and viabilityassessed by trypan blue dye exclusion. Analysis by light microscopyindicated that at least 90% of the cells were of the erythroid lineage.Aliquots of 4×10⁶ cells were transferred to individual microcentrifugetubes, pelleted, and resuspended in 1 ml of culture medium containing 11different dilutions of either rcEPO or rhEPO.

Changes in DNA synthesis were used to compare rcEPO to rhEPO in terms oftheir abilities to stimulate the replication of splenic erythroidprogenitor cells. Each erythropoietin dilution was evaluated intriplicate in a 96-well microtiter plate (Corning Science Products,Corning, N.Y.). The cells were plated at a density of 8×10⁵ cells in 0.2ml of culture medium. After 22 hours in culture, the cells were labeledfor 2 hours with 0.2 μCi³H-thymidiine (DuPont NEN, Boston, Mass.). Cellswere harvested onto glass filter mats using a Skatron cell harvester(Flow Laboratories. McLean, Va.) and ³H-thymidinie decay eventsquantified in a liquid scintillation counter (Beckman, Palo Alto,Calif.). Medium without erythropoietin supplementation and mediumconditioned by non-transfected CHO cells were used as negative controls.

The bioactivity of rcEPO was assessed in vivo by direct quantitation ofcirculating reticuloyctes in mice. Kawamura et al., “Simple in vivoBioassay for Erythropoietin,” Br. J. Haematol., 77:424-430 (1991).Normal adult C57BL/6J mice (The Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously for 3 successive days with either rcEPO orrhEPO at doses ranging from 0-20 units/mouse brought to a total volumeof 200 μl with PBS. Commercial rhEPO (Epogen®, Amgen, Inc., ThousandOaks, Calif.) was supplied at defined concentration of InternatinalUnits per mililiter (Storring et al., “The International Standard forRecombinant DNA-derived Erythropoietin: Collaborative Study of fourRecombinant DNA-derived Erythropoietins and Two Highly Purified HumanUrinary Erythropoietins,” J. Endrocinol., 134:459-484 (1992), which ishereby incorporated by reference). The amount of conditioned mediumcontaining equivalent units of rcEPO was estimated by Western Blotanalyses and in vitro bioactivity. Control mice received injections ofculture medium conditioned by non-transfected CHO cells. One day afterthe third injection, an aliquot of peripheral blood was collected intoEDTA-containing tubes. The percent of reticuloyctes in each blood samplewas determined by flow cytometric analyses of 10,000 cells using thefluorescent dye thiazole orange (Retic-COUNT, Bectin-Dickinson, SanJose, Calif.). Lee et al., “Thiazole Orange: A New Dye for ReticulocyteAnalysis,” Cytometry, 7:508-517 (1986); Nobes et al., “ReticulocyteCounting Using Flow Cytometry,” J. Clin. Pathol., 43:675-678 (1990),which are hereby incorporated by reference. Experimental protocols forboth murine bio-assays were reviewed and approved by the University'sInstitutional Animal Care and Use Committee.

Example 5 Statistical Analyses

For the in vivo bioassay, two-way analysis of variance was used todetermine the effect of recombinant erythropoietin source (canine orhuman), the dose of erythropoietin used, and whether there was anintraction between source and dose. When the interaction was notsignificant, data were interpreted for the main effects. The leastsignificant difference post hoc test was used to determine which dosesdiffered from each other and control. A value of p<0.05 was consideredsignificant.

Example 6 Results and Discussion

Canine EPO expression varied significantly between individualG418-resistant CHO cell clones co-transfected with pLEN-cEPO andpRSVneo. FIG. 19 illustrates comparative steady state levels of cEPO andEFTu mRNA in 100 individual clones. The clone used for subsequent rcEPOproduction was selected based on its high cEPO/EFTu mRNA ratio andexcellent growth characteristics. Western analysis of rcEPO protein inconditioned tissue culture medium identified a broad band ofapproximately 30-34 kDa, which was roughly equivalent in size tocommercial rhEPO (Epogen®, Amgen, Inc., Thousand Oaks, Calif., FIG. 3).Since erythropoietin is a glycosylated protein, enzymatic digestion ofrcEPO and rhEPO with N-glycosidase (FIG. 3, lanes 4 and 6) removed theheterogeneic carbohydrate residues and resulted in a single 18.5 kDabroad, a size consistent to that predicted by the primary cDNAnucleotide sequence.

Two assay systems were used to compare the bioactivity of rcEPO andrhEPO. In vitro, the replication of splenic erythroid progenitor cellswas stimulated in a dose-dependent manner by increasing levels of rcEPOsupplementation (FIG. 4A). No increase in 3H-thymidine incorporation wasobserved with culture medium conditioned by control CHO cells. The rcEPOstimulation exhibited an overall pattern of activity broadly parallel tocommercial rhEPO (Eopgen®, Amgen, Inc., Thousand Oaks, Calif., FIG. 4B).Similar bioactivity results were obtained using an in vivo murine assaybased on erythropoietin-induced stimulation with reticulocytosis.Untreated mice or mice injected with conditioned medium from control CHOcells had peripheral reticulocyte counts of approximately 3.5%. Thecounts increased significantly in a dose dependent fashion in responseto both rhEPO and rcEPO (FIG. 5).

Erythropoietin deficiency is the most important cause for thenonregenerative anemia that develops during chronic or acute renalfailure. It results from the loss of erythropoietin-producing cells inthe kidney during renal disease progression. This is true for bothhumans and companion animals. Recombinant hEPO became commerciallyavailable in 1989 and has dramatically improved the ability to managethis problem in humans. Exogenous erythropoietin replacement rapidlyrestores red blood cells mass and hemoglobin concentrations to normallevels and helps resolve many of the clinical symptoms associated withend-staoe renal disease. Therapeutic failure of rhEPO in companionanimals, estimated with an incidence between 20 and 50%, appears toresult from interspecies variation in erythropoietin structure. Althoughbiological activity of rhEPO is retained, the human protein isfrequently recognized as foreign by the immune system. The concept oferythropoietin replacement is appropriate for companion animals, theproblem is the immunogenicity of rhEPO. The pathogenesis and currenttreatment options for the anemia of chronic or acute renal failure indogs and cats have been reviewed by Cowgill, “Medical Management of theAnemia of Chronic Renal Failure,” In: Canine and Feline Nephrology andUrology, Osborne, et al., eds. Baltimore: William & Wilkins, 539-554(1995), which is hereby incorporated by reference.

The present invention demonstrates the feasibility of developingspecies-specific recombinant erythropoietin preparations. PCR was usedto amplify a canine erythropoietin cDNA fragment that was then used as aprobe to isolate phage clones containing the erythropoietin gene from acanine genomic library. DNA sequence analysis demonstrated 85.5%nucleotide identity with human erythropoietin over the full codingregion and predicts 81.3% identity at the amino acid level. Sequenceanalysis was carried out using the MacVector program with the defaultparameters. The 18.7% difference in primary amino acid sequence isconsistent with rhEPO being potentially immunogenic in dogs. IndividualCHO cell clones transfected with the pLEN-cEPO construct variety by morethan 100 fold in levels of rcEPO expression (FIG. 2). This likelyreflects differences in the site of integration and copy number of thetransgene incorporated. The size and apparent total glycosylation ofrcEPO was comparable to commercial rhEPO (FIG. 3). Importantly, the invitro (FIG. 4) and in vivo (FIG. 5) biological activity profiles ofrcEPO and rhEPO were similar in the two murine-based assays used in thisstudy. Combined, these results predict that rcEPO will stimulateerythropoiesis in dogs suffering from anemia secondary to an absolute orrelative deficiency of endogenous erythropoietin. Potential for animportant therapeutic advance is based on the prediction that use ofhomologouserythropoietin preparations will avoid the serious problems ofrhEPO's immunogenicity in companion animals.

Example 7 Purification of Recombinant Canine Ersythropoietin

rcEPO conditioned serum free tissue culture medium was used for clinicaltrial and bioactivity assays. An estimate of the rcEPO concentration indifferent batches of conditioned medium was determined by Western blotanalyses as illustrated in FIG. 3. For commercial use in companionanimals, however, the rcEPO must be purified and concentrated. Publishedmethods that have been used to purify human erythropoietin from theurine of sickel cell anemia patients should be applicable to thepurification of rcEPO from conditioned tissue culture medium. Miyake etal., “Purification of Human Erythropoietin,” J. Biol. Chem.,252:5558-5564 (1977); Krystal et al., “Purification of HumanErythropoietin to Homogeneity by a Rapid Five-Step Procedure.” Blood,67:71-79 (1986), which are hereby incorporated by reference. In fact,the process should be considerably easier since the startingconcentration of rcEPO is orders of magnitude higher than the level ofEPO in human urine and the CHO cells can now be cultured undercompletely serum free conditions.

In particular, the urine is filtered through a 0.45 gm membrane toremove cellular debris and other particulates. The filtrate is thenapplied to an ion exchange resin (CM Affi-Gel Blue, Pharmacia BiotechInc., Piscataway, N.J.) pre-equilibrated with 0.15 M NaCl and 10 mMNaPO⁴, pH 7.2. Bound proteins are eluted from the column with a steepsalt gradient from 0.15 M to 1.15 M NaCl in the same buffer (rcEPOelutes in a broad peak at approximately 0.9 M NaCl). the elute is thenconcentrated by ultrafiltration (amicon Model 402, Lexington, Mass.) anddialyzed overnight against 20 mM Tris-HCl, 0.1% PEG 4000, pH 7.0. Thesecond purification step involves chromatofocusing (PBE94column-polybuffer exchanger, Pharmacia Biotech) with a pH gradient from7.0 to 3.8 and back to 7.0. The pI of erythropoietin is reported to beapproximately 3.5 and the protein remains bound to the column. It isthen eluted with high salt (0.3 M NaCl in 20 mM Tris-HCl, pH 7.0) ratherthan lowering the pH of the eluent below erythropoietin's isoelectricpoint. The strategy of published methods diverge at this point. Krystalet al., “Purification of Human Erythropoietin to Homogeneity by a RapidFive-Step Procedure,” Blood, 67:71-79 (1986), which is herebyincorporated by reference, proceed with wheat germ lectinchromatography. The lectin, coupled to a Sepharose 6 MB macrobeadsupport (Pharmacia Biotech), binds N-acetyl-β-D-glucosaininyl and sialicacid residues in the carbohydrate side chains in erythropoietin.Non-glycosylated proteins wash through the column. Bound rcEPO is theneluted with 10 M N-acetyl-D-glucosamine in PBS containing 0.02% Tween20. In contrast, an earlier procedure (Miyake et al., “Purification ofHuman Erythropoietin.” J. Biol. Chem., 252:5558-5564 (1977) which ishereby incorporated by reference) utilizes Sephadex G-100 (PharmaciaBiotech) gel filtration chromatography. After concentration byultrafiltration, final purification steps employ reverse-phase HPLCusing either n-propanol or acetronitrile gradients.

Example 8 Materials and Methods for Examples 9-10

Erythropoietin preparations. Methods used in the production of rcEPOhave been described (MacLeod, et al., “Expression and Bioactivity ofRecombinant Canine Erythropoietin,” Am. J. Vet. Res., 59:1144-1148(1998), which is hereby incorporated by reference). Briefly, the geneencoding cEPO was isolated from a genomic library, subc-donded into aeucaryotic expression vector, and transfected into Chinese hamster ovary(CHO) cells (ATCC CRL-9618). A single high rcEPO-expressing CHO clonewas adapted for growth in defined serum-free medium^(a). Concentrationof rcEPO in conditioned medium was estimated by use of immunoblotanalyses and in vivo bioassasy (MacLeod, et al., “Expression andBioactivity of Recombinant Canine Erythropoietin,” Am. J. Vet. Res.,59:1144-1148 (1998), which is hereby incorporated by reference). Fortreatment of dogs, the concentration of rcEPO was normalized to 500U/ml, using medium conditioned by nontransfected control CHO cells as adiluent and supplemented with 0.25% (wt/vol) canine albumin and 0.025%(wt/vol) human albumin^(b). The rhEPO was purchased at a concentrationof 10,000 U/ml,^(c) diluted to a concentration of 500 U/ml with mediumconditioned by control CHO cells, and supplemented with canine and humanalbumin to final concentrations of 0.25 and 0.025%, respectively. Thediluent administered to dogs during weeks −4 to 0 consisted of mediumconditioned by control CHO cells and supplemented with canine and humanalbumin. The 3 preparations were sterilized by passage through 0.22 μmfilters and stored frozen at −20 C om sterile vials until used.

Dogs. Thirteen sexually intact clinically normal Baegels (9 females, 4ales) were studied for 28 weeks. Age of dogs was between 1.2 and 6.5years (median 1.3 years), and body weight was between 10.9 and 17.7 kg(mean, 13.7 kg) For 2 weeks prior to the start of the study, dogs wereacclimiated to environmental conditions and handling procedures. Alldogs were housed in same-sex groups of 2 or 3 and fed commercial dry dogfood, with water available ad libitum. Dogs were observed daily andcared for in a routine manner, including vaccination^(d) at the onset ofthe steady against canine distemper and adenovirus type-2, coronavirus,parvovirus, and leptospiral infections. The study was approved by theInstitutional Animal Care and Use Committee of Cornell University.

Dogs were randomly assigned to 2 groups; 1 group (dogs 1 to 6; 5females, 1 male) received rhEPO, and the second group (dogs 7 to 13; 4females, 3 males) received rcEPOc. Both groups received diluent, SC, 3times a week for 4 weeks (weeks −4 to 0) before initiating EPO (100U/kg) of body weight, SC 3 times/wk) treatment. This dosage of rhEPO hasbeen used by others to successfully stimulate erythropoiesis in dogs(Giger, “Erythropoietin and its Clinical Use,” Compend. Contin. Educ.Pract. Vet., 14:25-34 (1992); Cowgill, “Medical Management of the Anemiaof Chronic Renal Failure,” In: “Osborne Calif., Finco DR, eds. Canineand feline nephrology and urology. Baltimore: The Williams & WilkinsCo., 539-544 (1995), which are hereby incorporated by reference). Alldogs received supplemental iron (ferrous sulfate,^(e) 10 mg/kg, PO,daily or every other day) starting at the onset of EPO treatment.

During the steady, if Hct of any dog exceeded 65% for 2 consecutiveweeks, the EPO dose was decreased to 100 U/kg once weekly to avoidclinical problems associated with polycythemia. If dogs were receivingEPO at a dosage of 100 U/kg 3 times, and a >10% decrease from thehighest Hct determined after EPO treatment began was documented for 2consecutive weeks, EPO administration was stopped because of suspicionof erythroid hypoplasia. If dogs receiving EPO at a dosage of 100 U/kgonce weekly, and a >10% decrease from the highest Hct determined afterEPO treatment began was documented for 2 consecutive weeks, EPOadministration was restored to 100 U/kg 3 times weekly.

Laboratory assessments. Complete blood counts were performed monthly.Serum biochemical anlayses (sodium, potassium, chloride, total protein,albumin, globulin, BUN, creatine, glucose, calcium, phosphorus, totalbilirubin, cholesterol, and iron concentrations, alkaline phosphatase,aspartate transaminase, alanine, transaminase, γ-glutamyl-transferase,creatine kinase, and amylase activities, and unsaturated iron bindingcapacity) were done prior to initiating EPO treatment and at the end ofthe study, using automated procedures.^(f) Total iron-binding capacity(TIBC) was determined by summation of serum iron concentration andunsaturated iron-binding capacity. Transferrin saturation was calculatedas follows:

(serum iron concentration/TIBC)×100

Hematocrit (%) and RBC number (10⁶ μl) were determined weekly, usingautomated procedures.^(g) Reticulocyte count (%) was determined weeklyfrom new methylene blue-stained blood smears examined by use of oilimmersion light microscopy with the aid of a Miller's disk occuLar^(h)in 2 dogs and by use of flow cytometry^(i) in 11 dogs. Absolutereticulocyte count was calculated by multiplying the reticulocyte count(%) by the RBC count (10⁶/μl).

Bone marrow cytologic examination. Bone marrow aspirates were collectedfrom the wing of the ilium or proximal portion of the humerus asdescribed (Relford, “The Steps in Performing a Bone Marrow Aspirationand Core Biopsy,” Vet. Med., 86:670-688 (1991), which is herebyincorporated by reference), except that specimens were aspirated into asyringe containing 1 ml of citrate-phosphate-dextose solution. Bonemarrow smears were prepared from spiculated portions of the specimen andstained with modified Weight-giemsa.^(i) Myeloid-to-erythroid ratio(M:E) was determined from a 500 cell differential count. On the basis ofthe amount and intensity of stain updatek, iron stores were assessed onPrussian blue-tained bone marrow smears as absent, low, normal, orincreased. Bone marrow aspiration was performed before treatment withEPO (week 0) and at weeks 4, 8, 16 and 24. For the aspiration procedure,dogs were sedated with oxymorphone hydrochloride (0.05 to 0.10 mg/kg,1M) and midazolam (0.20 mg.kg, 1M).

Statistical analyses. The proportion of dogs developing from a 500 celldifferential count. On the basis of the amount erythroid hypoplasia inthe 2 groups was compared, using Fischer's exact test (Dawson-Saunders,et al., “Basic and Clinical Biostatistics,” Norwal, Conn.: Appleton andLange, 109-100, 114-116 (1994), which is hereby incorporated byreference). Hematologic and biochemical variables were compared beforeand after rcEPO or rhEPO treatment in each group, using the pairedt-test (Dawson-Saunders, et al., “Basic and Clinical Biostatistics,”Norwalk, Conn.: Appleton and Lange, 109-110, 114-116 (1994), which ishereby incorporated by reference). Differences in mean Hct, absolutereticulocyte count, leukocyte count, platelet number, and meancorpuscular volume (MCV) between rcEPO-treated dogs were evaluated at4-, 8-, 192-16, 20-, and 24-week time points, using Student's t-test(Dawson-Saunders, eta l., “Basic and Clinical Biostatistics.” Norwalk,Conn.: Appleton and Lange, 109-100, 114-116 (1994), which is herebyincorporated by reference). Values of P are reported with and withoutthe bonferroni correction for multiple comparisons (Dawson-Saunders, etal., “Basic and Clinical Biostatistics,” Norwalk, Conn.: Appleton andLange, 109-100, 114-116 (1994), which is hereby incorporated byreference).

Example 9 Hct and Absolute Reticulocyte Count in Dogs during EPOTreatment

Weekly mean Hct and absolute reticulocyte count increased in both groupsof dogs during the first 2 weeks of EPO treatment (FIG. 6). For dogsreceiving rcEPO, mean Hct continued to increase and, after week 4,exceeded the reference range (39 to 57%) throughout the study. Incontrast, mean Hct for dogs receiving rhEPO decreased after week 2 and,for weeks 13, 14, 15 and 17, was less than the reference range. (FIG.13A). Mean Hct in rhEPO- and rcEPO-treated dogs was significantly(P≦0.05) different for weeks 8 through 24. If a consecutive P<0.0078cutoff is used because of multiple comparisons. Hct for the 2 groupswere significantly different at weeks 20 and 24. Weekly mean absolutereticulocyte count peaked at 3 weeks of rcEPO treatment and exceeded thereference range (≦60,000 cells/μl) throughout the study (FIG. 13B). ForrhEPO-treated dogs, mean absolute reticulocyte count peaked after 2weeks, decreased to within the reference range for weeks 4 through 14;then, after rhEPO treatment had been discontinued in most dogs in thisgroup, increased to >60,000 cells/μl for weeks 15 through 19. Meanabsolute reticulocyte counts were significantly different between the 2groups for weeks 4 through 12 (P≦0.008) and week 24 P≦0.05).

Example 10 Erythroid Hypoplasia in Dogs Receiving rhEPO

All dogs (95% confidence interval, 63 to 100%) receiving rhEPO developederythroid hypoplasia, with M:E>15:1 (reference range, 0.75:1 to 2.5:1),by week 4 (n=4),8 (1), or 16 (1). In fact, 5 of these dogs had M:E≧49:1.With cessation of rhEPO treatment after diagnosis of erythroidhypoplasia, erythrocyte production recovered 5 to 11 weeks (median 7weeks) later in 5 of the 6 dogs (dogs 1 to 5). Dog 6 died of presumedanaphylaxis while still manifesting erythroid hypoplasia.

Unexpectedly, dog 1 redeveloped erythroid hypoplasia at week 24, eventhough treatment with rhEPO was reinstituted. In contrast, none (95%confidence interval, 0 to 37%) of the 7 dogs receiving rcEPO developederythroid hypoplasia during the study. In fact, during rcEPO treatment,all bone marrow specimens examined had erythroid hyperplasia, with M:Eof <0.75:1, except for dog 11 at week 16, which had cytologically normalbone marrow (M:E=0.96:1). However, that dog's rcEPO dosage had beenreduced to 100 U/kg/wk 6 weeks earlier between Hct was >65%: withreinstitution of 3-times-weekly rcEPO treatment, erythroid hyperplasia(M:E=0.61:1) redeveloped. The proportion of dogs developing erythroidhyperplasia was significantly (P≦0.01) different between the 2 groups.

Monthly mean leukocyte count was within the reference range (7.5 to19.9×10³ cells/μl) throughout the study for dogs receiving rcEPO.However, the rhEPO-treated dogs had mean leukocyte count below thereference range for weeks 8 through 16. (FIG. 2A). Differentialleukocyte count for weeks 8 through 16 in rhEPO-treated dogs indicatedneutropenia. Mean leukocyte count in rhEPO-treated dogs wassignificantly (P≦0.008) different from that in rcEPO-treated dogs forweeks 8 through 24. Monthly mean platelet count for rcEPO- orrhEPO-treated dogs varied but was within the reference range (1790 to510×10³ cells/μl) throughout the study (FIG. 2B). At week 16, meanplatelet counts for the 2 groups differed significantly (P=0.05).

Monthly mean MCV decreased in both groups of dogs between weeks 4 and 12(FIG. 2C). For dogs receiving rcEPO, means MCV continued to decreaseand, for weeks 16 through 24, was less than the reference range (64 to73 ff). In contrast, mean MCV in rhEPO-treated dogs increased for weeks16 through 24 after cessation of rhEPO treatment. Mean corpuscularvolume for the 2 groups differed significantly (P−0.004) at week 24.Mean corpuscular hemoglobulin concentration (MCHC) for both groups ofdogs remained within the reference range (31 to 37 g/dl).

Bone marrow iron stores were normal to increased in all dogs initially.However, 6 of 7 rcEPO-treated dogs and 2 of 6 rhEPO-treated dogs haddecreased marrow iron stores during periods of erythroid hyperplasia.Mean serum biochemical variable were within reference ranges at weeks 0and 24 for rhEPO- and rcEPO-treated dogs (Table 1). However, in eachgroup, specific significant differences was identified.

TABLE 1 Mean ± SEM values determined for serum biochemical variables atweeks 0 and 24 in clinically normal Beagles treated with recombinanthuman erythropoietin (rhEPO) or recombinant canine erythropoietin(rcEPO)^(x) rhEPO rcEPO Reference Week 0 Week 24 Week 0 Week 24 Variablerange (n = 6) (n = 5) (n = 7) (n = 7) Sodium (mEq/L) 141-156 146 ± 2 147 ± 4  146 ± 2  147 ± 4  Potassium (mEq/L) 3.8-5.5 4.6 ± 0.2 4.5 ± 0.24.6 ± 0.4   5.3 ± 0.5^(a) Chloride (mEq/L) 109-124 112 ± 1  111 ± 4  110± 3  109 ± 2  BUN (mg/dl)  8-30 16 ± 8  16 ± 4  25 ± 11 13 ± 4^(a ) Cr(mg/dl) 0.5-1.4 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1   0.6 ± 0.1^(a) Calcium(mg/dl)  7.2-12.8 9.8 ± 0.3 10.1 ± 0.2^(a ) 10.0 ± 0.3  10.4 ± 0.7 Phosphorus (mg/dl) 2.3-6.5 4.3 ± 0.4   4.0 ± 0.5^(a) 4.4 ± 1.1 4.7 ± 0.9TP (g/dl) 5.6-7.9 6.6 ± 0.6 6.6 ± 0.3 6.6 ± 0.5 7.1 ± 0.3 Albumin (g/dl)3.0-4.5 3.8 ± 0.2 3.8 ± 0.3 3.7 ± 0.2 3.7 ± 0.3 Globulin (g/dl) 1.8-4.22.8 ± 0.6 2.8 ± 0.5 2.9 ± 0.5   3.5 ± 0.5^(a) Glucose (mg/dl)  60-120 89± 12 92 ± 5  94 ± 20   74 ± 15^(a) ALT (U/L) 13-79 47 ± 19 35 ± 14 46 ±8  48 ± 15 AST (U/L) 13-52 24 ± 11 17 ± 5  21 ± 4  20 ± 4  ALP (U/L) 12-122 52 ± 21 55 ± 18 55 ± 25 70 ± 35 GGT (U/L)  0-10 8 ± 2  5 ± 2^(b)6 ± 1 5 ± 1 T Bili (mg/dl) 0.1-0.4 0.2 ± 0.1 0.1 ± 0.0 0.2 ± 0.1 0.2 ±0.0 Amylase (U/L)   454-1,380 754 ± 100   845 ± 156^(a) 792 ± 229   883± 247^(a,b) Chol (mg/dl) 124-335 139 ± 18  189 ± 39  189 ± 93    254 ±141^(a) CK (U/L)  58-241 219 ± 282   62 ± 17^(a) 99 ± 16 84 ± 23 Iron(μg/dl)  46-241 134 ± 23  148 ± 54  147 ± 31  159 ± 29  TIBC (μg/dl)235-495 363 ± 44  458 ± 92  432 ± 118 470 ± 92  % SAT (%) 17-69 37 ± 4 33 ± 11 35 ± 5  35 ± 9  ^(x)Starting at week 0, rhEPO or rcEPO wasadministered SC 3 times weekly at a dose of 100 U/kg of body weight.Frequency of erythropoietin administration was reduced in 4 rcEPO- and 1rhEPO-treated dogs because Hct increased to >65%. Treatment wasdiscontinued in all rhEPO-treated dogs no later than week 19 because ofdevelopment of erythroid hypoplasia. One rhEPO-treated dog died at week15; definitive cause of death was not determined. ^(a)Significant (P <0.05) difference between weeks 0 and 24 without adjustment for multiplecomparisons. ^(b)Significant (P < 0.002) difference between weeks 0 and24 with adjustment for 22 multiple comparisons. Cr = creatinine. TP =total protein. ALT = alanine transaminase. AST = aspartate transaminase.ALP = alkaline phosphatase. GGT = γ-glutamyltransferase. T Bili = totalbilirubin. Chol = cholesterol. CK = creatine kinase. TIBC = totaliron-binding capacity. % SAT = percentage of transferrin saturation.

Other than changes in hematologic variables, the clinical status of eachBeagle remained unchanged, with the exception of dogs 5, 6, 7, and 10.During the period of diluent administration, dogs 5 and 10 developedlethargy and fever of 1 days duration. Dog 5 was given an antibiotic(amoxicillin, 20 mg/kg, PO, q 12 h) for 5 days, but antibiotics were notgiven to dog 10, because the clinical signs of disease were less severe.Both dogs recovered without complications. Dog 7 treated with rcEPOdeveloped bacterial conjunctivities of the right eye that requiredtreatment with topically applied antibiotic ophthalmic ointment for theremainder of the study. After 15 weeks of study (4 weeks of diluent, 7weeks of rhEPO, and 4 weeks after rhEPO treatment was discontinued), dog7 died. Necropsy findings included acute diffuse severe pulmonarycongestion and edema without histologic evidence of cardiac lesions,suggestive of anaphylaxis. The precipitating cause of death was notdetermined.

Example 11 Recombinant cEPO Rescues a Dog From rhEOP-Induced Red CellAplisia

A dog with nonregenerative anemia secondary to chronic renal failure wastreated with rhEPO (100 units/kg, three times a week)(See FIG. 8). Thehematocrit rose for the first 6 weeks of therapy due to rhEPOstimulation of erythropoiesis. At approximately week 6 of therapy, thedog developed antibodies to rhEPO, blocking biological activity andinducing red cell plasia. From weeks 7 through 11, the hematocrit felldespite continued rhEPO therapy. At week 11, the hematocrit had droppedbelow 15% and a blood transfusion was given (arrow). Starting at week12, therapy was changed to rcEPO. Recombinant cEPO (00 units/kg, threetimes a week) restored red blood cell production, initially stopped afurther hematocrit decline and then stimulating a rise of hematocritback towards normal levels.

Results of this study indicate that rcEPO stimulates erythrocyteproduction in clinically normal Baegels's during 24 weeks of treatmentwithout the adverse effect of erythroid hypoplasia that was encounteredduring treatment of Beagles with rhEPO. Development of erythroidhypoplasia in rhEPO-treated dogs has been reported to coincide withappearance of antibodies against rhEPO (Cowgill, “Erythropoietin: ItsUse in the Treatment of Chronic Renal Failure in Dogs and Cats,” inProceedings Annu. Waltham/OSU Sump Treat Small Anim. Dis., 15:65-71(1991), which is hereby incorporated by reference). Seemingly, theseantibodies are elicited because of an 18.7% difference in primary aminoacid sequence between rhEPO and cEPO, rendering rhEPO potentiallyimmunogenic in dogs (MacLeod, et al., “Expression and Bioactivity ofRecombinant Canine Erythropoietin,” Am. J. Vet. Res. 59:1144-1148(1999), which is hereby incorporated by reference). In addition toblocking the efficacy of rhEPO, serocovnersion may result incross-neutralization of endoenous cEPO, resulting in erythroidhypoplasia. Cowgill, “Erythropoietin: Its Use in the Treatment ofChronic Renal Failure in Dogs and Cats,” in Proceedings Annu.Waltham/OSU Symp Treat Small Anim. Dis., 15:65-71 (1991); Giger,“Erythropoietin and Its Clinical Use.” Compend. contin. Educ. Pract.Vet., 14:25-34 (1992); Cowgill, et al., “Use of Recombinant HumanErythropoetin For Management of Anemia in Dots and Cats With RenalFailure,” J. Am. Vet. Med. Assoc., 212:521-528 (1998), which are hereinincorporated by reference). In rats, mice, and rabbits, monoclonalantibodies to rhEPO interfere with biological activity of rhEPO andendogenous EPO (Goto, et al., “Characterization and Use of MonoclonalAntibodies directed Against Human Erythropoietin That RecognizeDifferent Antigenic Determinants, Blood, 74:1415-1423 (1989), which ishereby incorporated by reference). Erythroid hypoplasia and anemia havealso been reported after administration of rhEPO to cats (Cowgill,“Erythropoietin: Its Use in the Treatment of Chronic Renal Failure inDogs and Cats,” in Proceedings Annu. Waltham/OSU Symp Treat Small Anim.Dis., 15:65-71 (1991); Cowgill, et al., “Use of Recombinant HumanErythropoietin For Management of Anemia in Dogs and Cats With RenalFailure,” J. Am. Vet. Med. Assoc., 212:512-528 (1998), which are herebyincorporated by reference) and horses (Woods, et al., “NonregenerativeAnemia Associated With Administration of Recombinant HumanErythropoietin to a Thoroughbred Racehorse,” Equine Vet. J., 29:326-328(1997); Piercy, et al., “Erythroid Hypoplasia and Anemia FollowingAdministration of Recombinant human Erythropoietin to Two Horse,” J. Am.Vet. Med. Assoc., 212:224-247 (1997), which are herein incorporated byreference). Data on the incidence of rhEPO-induced antibodies anderythroid hypoplasia in dogs are variable. In a study using dosages ofrhEPO comparable to those used in this study, 3 of 16 clinically normaldogs developed antibodies to rhEPO associated with progressiveanemia.^(k) In another report (Bader, “Stimulation of Bone Marrow byAdministration of Excessive Doses of Recombinant Human Erythropoietin,”Pathol. Res. Pract., 188:676-679 (1992), which is hereby incorporated byreference) in which rhEPO was administered IV to 15 Beagles at extremelyhigh dosages (100, 500, or 3,000 U/kg/d for 3 months), antibodyproduction was not detected except in dogs in the highest dosage group.In uremic does receiving rhEPO at dosages greater than those used inthis study, frequency of erythroid hypoplasia development approached 50%(Cowgill, Erythropoietin: Its Use in the Treatment of Chronic RenalFailure in Dogs and Cats,” in Proceeding's, Annu. Waltham/OSU Symp.Treat Small Anim. Dis., 15:65-71 (1991), which is hereby incorporated byreference). The higher frequency (100%, 95% confidence interval, 63 to100%) of erythroid hypoplasia encountered in the rhEPO-treatedclinically normal dogs of our study may reflect a breed or familialpredisposition to immunogenicity problems in our Beagle colony or achance observation.

The time of erythroid hypoplasia in rhEPO-treated uremic dogs has beenreported to be as early as 4 weeks after initiation of treatment, buttypically, it is between 10 and 13 weeks (Cowgill, “Erythropoietin: ItsUse in the Treatment of Chronic Renal Failure in Dogs and Cats,”Proceedings, Annu. Waltham/OSU Symp. Treat Small Anim. Dis., 15:65-71(1991), which is hereby incorporated by reference). In the rhEPO-treatedclinically normal dogs of our study, bone marrow erythroid hypoplasiaaccompanied by decreasing Hct and absolute reticulocyte count wasevident at 4 weeks in 4 of 6 dogs; the other 2 dogs developed erythroidhypoplasia by 8 and 16 weeks, respectively.

Recovery from rhEPO-induced erythroid hypoplasia after discontinuationof the dog has been described in dogs with chronic renal failure, butthe rapidity and extent of recovery are variable depending in part onthe diseased kidney's ability to produce adequate amounts of EPO(Cowgill, “Erythropoietin: Its Use in the Treatment of Chronic RenalFailure in Dogs and Cats,” in Proceedings, Annu. Waltham/OSU Symp. TreatSmall Anim. Dis, 15:65-71 (1991); Giger, “Erythropoietin and ItsClinical use,” Compend. Contin. Educ. Pract. Vet., 14:25-34 (1992);Cowgill, “Clinical Experience and Use of Recombinant HumanErythropoietin in Uremic Dogs and Cats,” in Proceedings, ACIVM Forum,9:147-149 (1991), which are hereby incorporated by reference). In theclinically normal dogs of this study, erythrocyte production recovered 5to 11 weeks (median, 7 weeks) after cessation of rhEPO treatment.However, after recovery, dog 1 redeveloped erythroid hypoplasia eventhrough rhEPO treatment was not reinstituted. It is interesting tospeculate whether anemnestic production of antibodies to rhEPOstimulated by the increase in endogenous. EPO may have resulted inrecurrence of erythroid hypoplasia.

Treatment with rhEPO and rcEPO initially stimulated erythrocyteproduction, as evidenced by increasing mean Hct and absolutereticulocyte count during the first 2 to 3 weeks (FIG. 6). However, withthe pressurized development of antibodies against rhEPO and resultingerythroid hypoplasia, mean absolute reciulatocyte count precipitouslydecreased, and mean Hct gradually decreased in rhEPO-treated dogs. Arebound increase in mean absolute reticulocyte count around week 16significant recovery from erythroid hypoplasia in most dogs once rhEPOtreatment was discontinued (FIG. 6B). In contrast, mean Hct forrcEPO-treated dogs continued to increase and, after week 4, exceeded thereference range (39 to 57%). In fact, 4 of 7 rcEPO-treated dogs requireda reduction in the frequency of EPO treatment (from 3 times weekly toonce weekly) because Hct exceeded 65%. Mean absolute reticulocyte countof >60,000 cells/μl and bone marrow cytologic findings of sustainederythroid hyperplasia during rcEPO treatment indicated continuedstimulated erythropoiesis.

In conclusion, the collective data from this study indicate that rcEPOstimulates erythrocyte production in clinically normal Beagles during a24-week treatment period without the adverse effect of erythroidhypoplasia commonly encountered in rhEPO-treated dogs. Recombinant cEPOappears to represent an improved option, compared with rhEPO, fortreatment of absolute or relative EPO deficiency in dogs.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose and variations can be made by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

1. An isolated nucleic acid molecule encoding a canine erythropoietin,wherein the canine erythropoietin has an the amino acid sequence of SEQID NO:3.
 2. The nucleic acid molecule of claim 1, wherein the nucleicacid molecule comprises bases 361-4491 of the nucleotide sequence of SEQID NO:1.
 3. The nucleic acid molecule of claim 1, wherein the nucleicacid molecule comprises the nucleotide sequence of SEQ ID NO:2.
 4. Anexpression vector comprising the nucleic acid molecule of claim 1,wherein the nucleic acid is foreign to the expression vector.
 5. Anexpression vector according to claim 4, wherein the expression vector ispLEN.
 6. An expression vector according to claim 6, wherein the nucleicacid molecule is inserted into the expression vector in proper senseorientation and correct reading frame.
 7. A host cell comprising theexpression vector of claim
 4. 8. The host cell according to claim 7,wherein the host-cell is a mammalian-cell.
 9. The host cell according toclaim 8, wherein the mammalian host cell is a Chinese hamster ovarycell.
 10. A host cell comprising the isolated nucleic acid molecule ofclaim 1, wherein the nucleic acid molecule is foreign to the host cell.11. An isolated canine erythropoietin protein or polypeptidedpolypeptide having an the amino acid sequence of SEQ ID NO:3.
 12. Theprotein or polypeptide according to claim 11, wherein the protein orpolypeptide is encoded by a nucleic acid molecule comprising bases361-4491 of the nucleotide of SEQ ID NO:1.
 13. The protein orpolypeptide according to claim 11, wherein the protein or polypeptide isencoded by a nucleic acid molecule having an the nucleotide sequence ofSEQ ID NO:2.
 14. A pharmaceutical composition comprising: a protein orpolypeptide according to claim 11 and a pharmaceutically acceptablediluent, adjuvant, or carrier.
 15. A method for providing erythropoietintherapy to a dog comprising: administering an effective amount of thepharmaceutical composition of claim 14 to the dog.
 16. A method forproviding erythropoietin therapy to a cat comprising: administering aneffective amount of the pharmaceutical composition of claim 14 to thecat.